Conventional techniques for cryopreservation of mammal cells are generally associated to disadvantages that decrease the potential use of these cells in the clinical and research fields.
Specifically, freezing spermatozoa is the most broadly used technique in assisted reproduction to preserve male gametes and provide the opportunity for future fecundation (Sanger et al., 1992). The main reported deterioration caused by cryopreservation in human spermatozoa is a marked reduction in motility (Critser et al., 1988). The primary cause of cellular damage produced during cryopreservation is caused by formation of intracellular ice crystals (Muldrew and McGann, 1990). This cellular damage involves the irreversible breakage of the plasmatic and nuclear membranes with a subsequent alteration of cellular organelles (Brotherton, 1990).
Traditional spermatozoa freezing methods use intracellular cryoprotectants because they have the ability to permeate the plasmatic membrane and are able to penetrate the cell. The efficiency associated to sperm function using this type of cryoprotectants is low, with at most 45% of conservation of at least one relevant cell function that is important for this type of cells, such as motility, because permeable cryoprotectants are cryotoxic and exert damage to intracellular organelles and the plasmatic membrane, notably decreasing the fecundation capacity of spermatozoa. Furthermore, this technique has a high cost due to the type of media used and the prolonged preservation in liquid N2, and therefore its use is limited to higher income populations or to programs with costs that are not covered by the health system, as in developed countries.
Vitrification is an extremely fast cryopreservation method that requires less execution time, is safer and has a lower effective cost than traditional freezing. Vitrification has been widely investigated for embryos and oocites (Chen et al., 2001; Isachenko et al., 2005), but is much undeveloped for spermatozoa, and few publications about this topic can be found (Isachenko et al., 2004; Isachenko et al., 2008). In these last publications, the authors, who are also the inventors of the present invention, developed the basis for a cryopreservation method for spermatozoa, free from toxic cryopreservants. However, this methodology is not aseptic and does not have the characteristics of portability, simplicity and efficacy, which are indeed provided by the kit and optimal composition of the media of the present invention.
This method is similar to glass formation, which is an intrinsic property of all liquids that only needs a sufficiently fast freezing rate to avoid the passage through the crystalline stage. For this reason, vitrification does not cause protein molecules to approach to each other as in traditional freezing (Levitt, 1966), which avoids formation of disulfide bridges that cause protein denaturation (Fahy et al., 1984).
Vitrification is carried out in small volumes in such a way as to immerse the sample directly in liquid nitrogen to increase the cooling rate and decrease ice crystal formation (Nawroth et al., 2002). This contributes even more to facilitate the methodology and costs of spermatozoa vitrification, since it does not require special cryobiological equipment. Likewise, it allows adequate preservation of sperm motility, but the most significative advance of this technique is that it does not damage sperm DNA.
The Patent Application US20080026361, filed in 2007, discloses a composition comprising a cryoprotectant, a protective membrane that stabilizes or assists in stabilizing spermatozoal membranes and a radical scavenger, wherein the cryprotectant is a sugar, such as sacarose, the protective membrane can be a protein agent or a non-protein agent or a combination of the same, albumin being an example of a possible protein agent, and the radical scavenger can be a reducing agent or an antioxidant. This application claims a kit that comprises a vitrification composition, an insulating container, a cryogenic pack, a receptacle for spermatozoa, such as straws, glass ampoules, cryotubes or cryovials. However, the application uses a cryoprotectant solution with a different composition to that of the present invention; using a medium mainly containing glycerol (TYB) and optionally supplemented with monothioglycerol.
The Patent Application US20090305224 (family of WO2007120829), filed on 2009, describes a method for cryopreservation of mammal cells such as oocites, hepatocytes, stem cells, embryos or cygotes, useful as a clinical or research tool (e.g. in reproductive technology, cell transplant, tissue engineering or regenerative medicine). The application focuses on the determination of the thermal performance parameters of different materials and solutions, such as thermal conductivity of diverse materials, mainly quartz. Moreover, this patent application does not exemplify the use of the proposed method for specific vitrification of human spermatozoa and does not exemplify the straw materials of the present invention, since the use of quartz not only increases the associated costs, but also restricts the portability and applicability of thermal conductors of this material in a commercial kit.
The article “Impact of three different cryoprotectants on the motility of post-thaw human sperm” (Hu et al., 2007) is aimed at comparing the effect levels of a glucose cryoprotectant or cryoprotectants with different sucrose concentrations on the motiliy of human spermatozoa after thawing. In the study, no large differences with other cryoprotectants were observed when using glucose as a cryoprotectant at a concentration of 0.1 M. However, when using sucrose at a concentration of 0.2 M, a significant decrease in spermatozoal damage was observed in comparison with other cryoprotectants. Surprisingly, using of a sucrose concentration in the range higher than 0.2 M constitute an optimal concentration for the spermatozoa vitrification medium of the method of the present invention.
The article “Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose” (Isachencko et al., 2008) investigates the capacity of sucrose as a cryoprotectant for spermatozoa against mitochondrial damage, and its effect on cryocapacitation and acrosomal reaction during vitrification. The procedure includes the selection of spermatozoa using different culture media, where one particular medium comprises HTF (Human Tubal Fluid) supplemented with 0.25 M sucrose and 1% HSA (Human Serum Albumin). After vitrification, different spermatozoal functional properties were evaluated, and the article concludes that the medium with sucrose was significantly better to preserve spermatozoal progressive motility after devitrification. The methodology that has been used to vitrify the cell suspension consisted in vitrifying 30 μL spheres that were dropped directly into liquid nitrogen, this being an effective but not aseptic methodology, since droplets directly contacted the liquid nitrogen. Surprisingly, the use of the method of the present invention, besides being aseptic, allows the preservation of a volume up to 100 μL, and thus is a more efficient method when larger sample volumes are available.